Seed borne diseases have a devastating economic effect on world agriculture on a yearly basis. Accurate information on crop losses is difficult to obtain due to the lack of an organized effort worldwide to assimilate such data. However, a publication was located which stated that the total annual world crop losses due to seed borne diseases amounts to the total crops producing in South America in a year (Farm Economy, 1978, Volume 1, Issue 1, Bangladesh Agricultural Economists' Association). A more specific example to show on a small scale the scope of the problem of seed borne diseases can be found in a single crop in a relatively small country like Israel. In the year 2013 due to a seed borne disease in the form of the virus CGMMV in watermelons caused an estimated US$20 Million in crop losses. Furthermore, indirect losses will occur in years to come due to the wide spread contamination of crop land that cannot be used for growing watermelons in a country already severely limited due to the lack of space.
In addition to economic damage in agriculture, seed borne pathogens such as E. coli have been implicated in human health diseases (National Institute of Allergy and Infectious Diseases website-accessed Jan. 2, 2014). Seed borne diseases come in a variety of forms including fungi, bacteria, viruses, and viroids. Seed borne diseases affect nearly all agricultural seed crops including vegetables, flowers, cereals, legumes, and field crops. Inoculum of seed borne diseases can be found on the surface of the seed. In addition, inoculum can be found internally in the testa/pericarp, endosperm/cotyledons, and the embryonic axis (Roberts, S J, 2009, Tropical Plant Pathology).
Recently, due to the globalization of the seed industry, seeds are produced in many parts of the world and then sold to a wide range of countries. Therefore, the potential for spreading diseases via seed is great. To date many methods have been developed for disinfecting seeds, but all are far from ideal. Most are only effective for external infections. The few available that are effective for internal infections commonly cause germination problems. Thus, seed borne diseases have become a widespread problem.
The various types of treatments available can be categorized as chemical, physical, and biological. Chemical treatment entails introducing various chemicals including resistance inducers, synthetic, natural, organic, inorganic, systemic, and non-systemic chemicals by various means. Physical treatment includes use of hot water, hot air, steam, dry heat, ultrasound, and radiation. Biological treatments entail the introduction of biological agents that either eradicate the disease or induce natural resistance.
Physical treatments are difficult to use as there is a fine line between effectively eradicating seed borne disease and injuring seeds. Not all batches of seeds react the same to all treatments, and therefore it is difficult to predict how physical treatments will affect seed germination and vigor. Chemical treatments are available that have no phytotoxic effects on seeds, but methods to get complete internal penetration have not been identified.
The use of ultrasound, irradiation, and a soak for up to 60 minutes with acidic electrolyzed water for the eradication of the bacteria Escherichia coli O157:H7 on alfalfa and broccoli seeds was examined (Kim, H Y et al, 2006, Journal of Food Science Vol. 71, Nr. 6). The study determined that none of these methods were able to deliver satisfactory results. Ultrasound and irradiation are physical treatments, while a 60 minute soak with electrolyzed water is a chemical treatment.
A method of eradicating Acidovorax avenae subsp. citrulli from melon and watermelon seeds by soaking seeds in various chemicals has been taught (Feng, J. et al, 2009, Canadian Journal of Plant Pathology, volume 31, pages 180-185). The study claims success in eradicating Acidovorax avenae subsp. citrulli from triploid watermelon seeds using a 30 minute soak with electrolyzed water. However, the design of the study was flawed in that infected seeds used in the study were diploid watermelon seeds, and not triploid seeds. Triploid watermelon seeds used were healthy, and were merely mixed with infected diploid seeds. This only proves that the method is effective on diploid watermelon seeds. In addition, infected seeds were rinsed with a 0.5% sodium hypochlorite solution and therefore assumed to be free from external inoculum. While a 0.5% sodium hypochlorite solution is effective on smooth surfaces such a plastic, on seed it has not been demonstrated to be effective. It was demonstrated that a 1% calcium hypochlorite solution used as a soak for 15 minutes was not effective in eliminating Acidovorax avenae subsp. citrulli from watermelon seeds (Hopkins, D. L. and Cuccuuza J. D., 1996, Wet Seed Treatments for the Control of Bacterial Fruit Blotch of Watermelon, The American Phytopathological Society, publication no. D-1996-0223-05R). Calcium hypochlorite has greater available chlorine than sodium hypochlorite and is therefore a stronger disinfectant. Therefore, the ability to disinfect seeds internally and the ability to disinfect triploid watermelon seeds remained unproven.
Seed priming is a term used in the seed industry to describe a pre-sowing hydration treatment developed to improve seedling establishment. Seed priming has a long history of use for increasing seed germination in vigor. For example, research is on record as early as 1943 in the field of seed priming (Levitt, J. and Harem, P. C., 1943, A method of increasing the rate of seed germination of Taraxacum kok-saghyz. Plant Physiology 18(2): 288-293). Seed priming techniques can be divided in to three main categories which are osmopriming (osmoconditioning), hydropriming (drum priming), and matrix priming (matriconditioning).
The goal with all three mentioned types of priming is to allow a controlled imbibition and induction of the pre-germinative metabolism (“activation”), but radicle emergence is prevented. The hydration treatment is stopped before desiccation tolerance is lost. An important part of the priming process is to stop the process in the right moment, which varies depending on the species and the seed batch. With osmopriming, seeds are incubated in well aerated solutions with a low water potential, and afterwards washed and dried. The low water potential of the solutions can be achieved by adding osmotica like mannitol, polyethyleneglycol (PEG) or salts like KCl. Hydropriming is achieved by continuous or successive addition of a limited amount of water to the seeds. A drum is used for this purpose and the water can also be applied by humid air. On-farm steeping is a simple and useful form of hydropriming that is practiced by incubating seeds for a limited time in water. Matrix priming is the incubation of seeds in a solid, insoluble matrix (vermiculite, diatomaceous earth, calcined clay, and water-absorbent polymers) with a limited amount of water. This method confers a slow imbibition.
A method of combining matrix priming with chemical or biological treatment was suggested (Eastin, J. A., 2000, U.S. Pat. No. 6,076,301). This method suggests that chemicals or microbes can be mixed with water used for priming, thereby the seeds imbibe the chemical or microbes. This method can be used as a technique both for disinfecting seeds with chemicals, or impregnating seeds with biological agents.
The effectiveness of using an antibiotic solution combined with matrix priming for disinfecting triploid watermelon seeds internally infected with Acidovorax avenae subsp. citrulli has been tested. In order to screen for effective antibiotics, diploid watermelon seeds were obtained which were externally infected with Acidovorax avenae subsp. citrulli. Various antibiotics were tested including Kasugamycin, Oxytetracycline, Chloramphenicol, Oxalinic acid, Ciprofloxacin, Ceftriaxone sodium, Trimethoprim-sulfamethoxazole, Colistin sulphate, and Piperacillin/tazobactam. Infected seeds were soaked for 10 minutes in antibiotic solutions of 100 ppm, 250 ppm, and 500 ppm of the above antibiotics. Dosage was chosen based on information reported by Layne Wade (21st Annual Tomato Disease Workshop, Asheville, N.C., November 2006) which stated that the MIC (minimum inhibitory concentration) of Kasugamycin on Acidovorax avenae subsp. citrulli is greater than 100 ppm. Results were as expected in that 100 ppm was not sufficient to eliminate bacteria on seeds for any of the above antibiotics. Kasugamycin, Oxytetracycline, Chloramphenicol, Oxalinic acid, Trimethoprim-sulfamethoxazole, Colistin sulphate, and Piperacillin/tazobactam were all effective at both 250 ppm, and 500 ppm. Ciprofloxacin and Ceftriaxone sodium were not effective at any dose. One of the effective antibiotics was chosen which was Oxytetracycline at 500 ppm and triploid watermelon seeds were matrix primed with this solution for 6 days. Bacteria in the seeds were not eliminated using this method.
A method of vacuum infiltration of seeds was taught for the purpose of impregnating seeds with beneficial bacteria (Porter, F. E. and McAlpine, V. W., 1960, U.S. Pat. No. 2,932,128). Vacuum infiltration is the process of putting seeds mixed with a liquid medium such as water mixed with chemicals or other substances, in a state of reduced pressure in a vacuum chamber. This method was developed in order to eliminate the need for soaking seeds in a solution containing bacteria or other microorganisms for extended periods of time. Extended soaking can start the germination process in as little as 2 hours, and therefore seeds would have to be planted within 24 hours, and there is no opportunity to dry back the seeds for storage and future use. This method allowed sufficient penetration with only 10 minutes of soaking because of the state of reduced pressure. This method of using vacuum infiltration was used to obtain deep penetration of antibiotics as well as other chemicals for the treatment of melon seeds infected with Acidovorax avenae subsp. citrulli (Frare, V. C., 2010, Melon (Cucumis melo L.) seeds treatment to control Acidovorax avenae subsp. citrulli, Luiz de Queiroz College of Agriculture). However, no chemical treatment was successful in eradicating the disease from infected seeds using vacuum infiltration.
Upon investigation, it was determined that the problem with the vacuum infiltration not working may have been due to the treatment time of 10 minutes not being long enough. Therefore, testing vacuum infiltration with oxytetracycline at 500 ppm for an extended period of time beyond 10 minutes was carried out. It was expected that by doubling or tripling the 10 minute treatment period, disease would be eradicated from infected seeds. To cover the extreme case, a treatment period 10 times the 10 minute period was done in addition to double and triple. Surprisingly, not even the 100 minute treatment successfully eradicated the seed borne disease.
Following this unsuccessful attempt with vacuum infiltration, it was further surmised that the concentration of antibiotic needed to be increased, and that the treatment length was not the problem. Therefore, a further attempt was made to eradicate seed borne disease using vacuum infiltration with both an extended treatment period, and high concentrations of antibiotic. While previously it was expected that 500 ppm of oxytetracycline would be sufficient, this concentration was doubled, tripled, and for an extreme case to 10 times the original concentration equal to 5000 ppm. Infected seeds were treated with these antibiotic concentrations for 10 minutes, 20 minutes, 30 minutes, and for the extreme case 100 minutes. By accident the 10 minute treatment was forgotten and left for more than 24 hours under vacuum infiltration. Results after laboratory analysis showed that only the treatment for more than 24 hours was effective.
Following the success of the extreme length of vacuum infiltration of 24 hours at a high antibiotic concentration, a low concentration of antibiotics (500 ppm) was then tested for an extreme length of vacuum infiltration of 24 hours. Seed borne disease was eliminated showing that the lengthy treatment with vacuum infiltration and not the high concentration of antibiotics was the key to an effective treatment.
Experiments on other vegetable crops, including broccoli, kohlrabi, watermelon, melon, tomato, pepper, and carrots, have concluded that the minimum time needed under vacuum infiltration in an antibiotic solution to effectively reduce seed borne disease was approximately two hours. However, treating a vast array of agriculture seeds for more than two hours in an aqueous solution commonly causes a decrease in seed quality. This makes the use of a treatment of vacuum infiltration with an aqueous solution for more than two hours not a viable solution for eliminating seed borne diseases in agricultural seeds. Thus, a possible solution to this problem is to combine a method of seed priming together with the lengthy vacuum infiltration in aqueous solutions.
However, to date, these methods have not been combined successfully to achieve superior results.